Microplate-reader with a controlled gas atmosphere, corresponding method and use of same

ABSTRACT

A microplate reader and method has at least one measuring device and a holding device for accommodating at least one microplate and for positioning samples-containing wells of microplates in relation to the measuring device. The at least one measuring device is used for detecting light emitted by samples in wells of a microplate and/or which is influenced by samples transilluminated by light in the wells. The microplate reader has a control unit for controlling the composition of a gas atmosphere surrounding the wells containing the samples. A respective use is characterized particularly in that living cells are measured in a controlled gas atmosphere, wherein the living cells are chosen from microaerophilic, optionally anaerobic and obligatorily anaerobic microorganisms as well as fungi and eukaryotic cells.

RELATED APPLICATIONS

This patent application claims priority of the U.S. provisional application No. 61/380,783 of Sep. 8, 2010, the disclosure of which is incorporated herein by reference in its entirety for any purpose.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a microplate reader which comprises at least one measuring device and one holding device. The at least one measuring device is used for the detection of light which is emitted by samples in wells of a microplate inserted in said microplate reader and/or which is influenced by samples penetrated by light in wells of a microplate inserted in said microplate reader. The holding device is used for accommodating at least one microplate and for positioning the wells of said microplate(s) containing the samples in relation to the at least one measuring device. The invention further relates to a respective method and special uses of such microplate readers.

RELATED PRIOR ART

Generic microplate readers which have been known for many years in the state of the art are based on the principle of the measurement of luminescence and/or fluorescence of samples treated with a reagent. Luminescence or fluorescence generally refers to the emission of light which originates from a sample, wherein the luminescence is caused by the progression of a chemical reaction in a sample and the fluorescence is caused by the irradiation of an excitation light. Such microplate-readers are used for observing reactions in samples to which a reagent is added or for detecting specific sample components which can be made to fluoresce.

Respective microplate readers are known which—for the purpose of triggering a luminescent reaction—comprise an injector apparatus for adding a reagent to the samples in wells of the microplate(s) used in respectively inserted into this microplate reader. Microplate readers are also known which comprise an illumination device for irradiating or transilluminating samples in wells of the microplate(s) used in this microplate reader. Such microplate readers are used to measure the fluorescence excited in the samples or the reduction in the transparency (the absorbance) caused by the samples.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to propose a microplate reader, respective methods for measuring living cells in a microplate reader and uses of such a microplate reader which enable the observation of reactions in samples and/or the detection of specific sample components under at least approximately physiological conditions.

This object is achieved with respect to a first aspect by a microplate reader as herein disclosed. This microplate reader comprises:

-   a) at least one measuring device, which is selected from a group     comprising:     -   a1) a first measurement device which is designed for measuring         the absorbance of samples in wells of a microplate used or         inserted in the microplate-reader,     -   a2) a second measurement device which is designed for measuring         the fluorescence of samples that are irradiated with light in         wells of a microplate used or inserted in the microplate-reader,         and     -   a3) a third measurement device which is designed for measuring         the luminescence of samples in wells of a microplate used or         inserted in the microplate-reader; -   b) a holding device for accommodating at least one microplate and     for positioning the samples-containing wells of these microplate(s)     in relation to at least one measuring device; -   c) a control unit for controlling the composition of the gas     atmosphere surrounding the samples-containing wells of microplates     used or inserted in this microplate reader.

The microplate-reader further comprises alternatively:

-   d1) a separating plate which subdivides an interior space of a     housing of the microplate-reader into an appliance compartment and a     sample compartment and which comprises at least one opening, which     separating plate is designed such that light which is irradiated or     influenced by samples in wells of a microplate used in the     microplate-reader can pass therethrough from the samples compartment     to the appliance compartment, or -   d2) an interior housing, which is arranged within a housing of the     microplate-reader, within which the holding device is arranged,     which interior housing subdivides an interior space of the housing     of the microplate-reader into an appliance compartment and a sample     compartment surrounding the holding device, and which comprises at     least one opening, which is designed such that light which is     irradiated or influenced by samples in wells of a microplate used in     the microplate-reader can pass therethrough from the samples     compartment to the appliance compartment.

A main function of the separating plate and of the interior housing is common to these two alternative zoning means, i.e. separating of the sample compartment from the appliance compartment in a way that the samples in the sample compartment are influenced by the appliances only as desired.

In the interior space of the housing, the microplate-reader can be designed such that the sample compartment is separated from the appliance compartment substantially light-tight and substantially gas-tight by means of the separating plate respectively the interior housing. To this end, the opening may comprise an according proofing device. Alternatively or in addition, a proofing device which extends around the opening may be provided. In the embodiments described, the proofing device may be designed such that it influences primarily substantially the passing of light through the opening, namely such that the light which is has been emitted or influenced by samples in wells of a microplate used in the microplate-reader may pass through the opening from the sample compartment to the appliance compartment, while other light (which has not been emitted or influenced by the samples) is blocked as completely as possible from passing through the opening.

A movement device for mixing and/or circulating gas that is present in and/or flowing into the sample compartment may be arranged in the sample compartment.

The movement device may comprise at least one of the following devices: a blower, a device comprising one or more baffle plates, an agitation device comprising one or more paddles, or a structured jet nozzle system. Thereby, the device comprising the one or more baffle plates may be arranged near by a gas inlet into the sample compartment. The agitation device comprising the one or more paddles may be arranged near by the holding device. The structured jet nozzle system may comprise a plurality of inlet nozzles, which are arranged substantially uniformly distributed in the sample compartment, so as to effect that during the inflow of gas through the inlet nozzles the gas atmosphere in the sample compartment is moved and mixed substantially uniformly. The inlet nozzles may be arranged to be distributed, preferably equally spaced, along a gas inlet line. The gas inlet line may sneak in an arrangement substantially as whole along respectively substantially parallel to a wall surface, such as ceiling or bottom wall surface, of the sample compartment. The arrangement may comprise a substantially S-shaped arrangement, a plurality of substantially S-shaped arrangements and/or a substantially spiral-shaped arrangement. Alternatively or in addition, the gas inlet line may divide or bifurcate at least in sections, in one or plural sections, into two or more gas inlet sub-lines.

The control unit may comprise a computer having, for example stored therein, corresponding software, wherein said computer may be connectable to a central computer of the microplate-reader or integrated therein. Alternatively, the control unit may comprise a computer having corresponding software, wherein this computer is connectable to a central computer of the microplate-reader and arranged in a separate housing.

The control unit may be designed to control the composition of the gas atmosphere comprising up to four, five, six or more different gases. To this end, the control unit may comprise gas sensors for measuring different gases in the sample compartment, i.e. for controlling the composition of gas atmosphere surrounding the samples-containing wells of a microplate used in this microplate-reader. Independent from this, the microplate-reader may comprise a gas inlet, in particular a gas inlet without gas sensor, which inlet is actuatable by the control unit and which is for admitting gas into the sample compartment.

At least one gas of the different gases may be selected from a group which comprises nitrogen (N₂), carbon dioxide (CO₂), oxygen (O₂), carbon monoxide (CO), hydrogen sulphide (H₂S) and sulphur dioxide (SO₂).

The control unit preferably comprises an O₂-sensor for measuring and controlling the oxygen content of the gas atmosphere surrounding the samples-containing wells of a microplate used in this microplate-reader and a CO₂-sensor for measuring and controlling the carbon dioxide content of the gas atmosphere surrounding the samples-containing wells of a microplate used in this microplate-reader.

Independent from this and/or further to this, the control unit may be designed to control the humidity and the temperature of the gas atmosphere. To this end, the control unit may comprise in the sample compartment a humidity sensor for measuring the humidity of the gas atmosphere and a temperature sensor for measuring the temperature of the gas atmosphere.

Independent from this and/or further to this, the control unit may comprise a cooling device for cooling and/or a heating device for heating the sample compartment. Preferably, the heating device is thereby mounted on the separating plate or on the interior housing respectively and is in heat exchange communication with the gas atmosphere in the sample compartment, and the cooling device is mounted on the bottom of the sample compartment and is in heat exchange communication with the gas atmosphere in the sample compartment. The heating device may be mounted on a plate which is arranged on the sample compartment side of the separating plate resp. on the side of the interior space of the interior housing. Preferably, the plate is connected non-heat conductingly with the separating plate resp. with the interior housing. A condensation of humidity respectively water on the microplate respectively at, on and/or in the wells of the microplate is counteracted by these arrangements of the heating device and the cooling device. What has been disclosed here with respect to the cooling device resp. the heating device applies likewise for the design of the microplate-reader having a separating plate resp. having an interior housing.

A separate ventilation device, which may be arranged in the appliance compartment, may be provided in the microplate-reader. The ventilation device serves for cooling light sources (e.g. lamps) and/or other devices which generate heat. The ventilation device may be actuated respectively operated independently from the heating device arranged in the sample compartment respectively the cooling device arranged in the sample compartment.

In the microplate-reader, the first measurement device, the second measurement device and/or the third measurement device may respectively comprise a light guide having an admission point for light and an exit point for light, and a light detector device arranged for measuring light exiting from the exit point. The first accession point for light into the first measurement device, the second accession point for light into the second measurement device and the third accession point for light into the third measurement device may respectively be arranged in the sample compartment.

Herein, the term light guide is understood to refer to an optical fiber, to an optical fiber bundle, or to a mirror system. Light guides embodied as an optical fiber or an optical fiber bundle, and a light guide embodied as a mirror system comprise an admittance point for light, an exit point for light and a light guide passage, which extends from the admittance point to the exit point and along which light can be guided by the light guide.

In the microplate-reader, a respective light guide of the first, second and/or third measurement device may be embodied as an optical fiber or an optical fiber bundle. Alternatively, a respective light guide can be embodied as a mirror system. Combinations of optical fibers and mirrors are feasible for the illumination of the samples as well as for the detection of the light coming from the samples. The light detector device of a respective first, second and/or third measurement device may be arranged in the sample compartment or in the appliance compartment.

In the microplate-reader, the first light detector device of the first measurement device and/or the third light detector device of the third measurement device may be arranged in the sample compartment. In the microplate-reader, the second light detector device of the second measurement device designed for measuring the fluorescence in wells may be arranged in the appliance compartment.

The microplate-reader may comprise at least one illumination device designed for transilluminating respectively for irradiating samples in wells of a microplate used in the microplate-reader with light. The illumination device may comprise a light source and a light guide for guiding at least a portion of the light generated by the light source to resp. into a well of a microplate used in the microplate-reader respectively to a sample arranged in a well.

A first, second and/or third illumination device may be provided, respectively, for a respective first, second and/or third measurement device. However, it also possible to provide that an illumination device is provided in common for the first and second measurement device, the second and third measurement device, the first and third measurement device respectively in common for the first, second and third measurement device.

A respective light source of an illumination device may be selected from a group, which comprises a laser, a flash lamp, an LED (light emitting diode) and a laser diode.

In particular uses of the microplate-reader, it may be important to measure resp. to detect kinetics of the growth resp. the transformation of cell cultures which are arranged as samples in a well of a microplate. The measuring resp. detecting of the kinetics may be achieved by measuring resp. detecting, in particular a change as a function of time, of the absorbance, the fluorescence and/or the luminescence of the cell culture (sample), preferably the luminescence of the cell culture (sample).

For measuring resp. detecting kinetics that proceed temporally relatively quickly, it may be necessary, in particular advantageously, that the light source of the illumination device generates relatively short light pulses. Therefore, a pulsed laser, a flash lamp, an LED (light emitting diode) operated in a pulsed mode, and/or a laser diode operated in a pulsed mode may be light source suitably for this purpose.

A flash lamp which is not actively cooled may be provided in the microplate-reader as a light source. This flash lamp may be arranged in the appliance compartment. Further, light generated from this light source may be guided by a light guide to a sample, which as arranged in a well of a microplate used in the microplate-reader, for irradiating and/or transilluminating the sample.

The microplate-reader may comprise a first illumination device, which is designed for transilluminating samples in wells of a microplate used in the microplate-reader with light and which is arranged in the appliance compartment. Thereby, the first measurement device may be arranged in the sample compartment. The second measurement device may be arranged in the appliance compartment. The third measurement device may be arranged in the sample compartment or in the appliance compartment. Thereby, the first accession point of light into the first measurement device, the second accession point of light into the second measurement device and the third accession point of light into the third measurement device may be arranged in the sample compartment.

The microplate-reader may comprise a first illumination device, which is designed for transilluminating samples in wells of a microplate used in the microplate-reader with light. Thereby, an exit point of the first illumination device may be arranged in the sample compartment. The microplate-reader may further comprise a second illumination device, which is arranged in the appliance compartment and which is designed for irradiating samples in wells of a microplate used in the microplate-reader with light. Thereby, an exit point of the first illumination device may be arranged in the sample compartment. The second illumination device may be selected from the group, which comprises a laser, a flash lamp, an LED (light emitting diode) and a laser diode.

The first, second and/or third measurement device may comprise a first, second and/or third optical system, wherein at least one of these optical systems and/or a used (inserted) microplate are designed to be movable relative to each other in the direction of a Z-axis of a Cartesian coordinate system. Thereby, the used (inserted) microplate may be designed to be movable in the direction of the Z-axis. Alternatively resp. in addition to this, the first, second and/or third optical system may be designed to be movable in the direction of the Z-axis. Preferably, the first, second and/or third optical system is designed so that it can be lowered partly into the sample compartment through the at least one opening in the separating plate resp. in a wall, such as a ceiling wall, of the interior housing.

In the corresponding method for measuring living cells in a microplate-reader (see below) and/or in the use of a microplate-reader (see below) it may occur that a reagent is added, e.g. using an injector device, to at least one sample in at least one well of the microplate and triggers a chemical reaction. With the triggering of the chemical reaction, there may go along a luminescence reaction resp. a change of the luminescence characteristics, a change of the fluorescence and/or a change of the absorbance of the sample. These may be detected, e.g. using at least one of the measurement devices, for example by observing (measuring resp. detecting) the luminescence and/or the fluorescence and/or the absorbance of the sample.

For adding a reagent, the microplate-reader may comprise an according injector apparatus designed for adding a reagent to the samples in wells of a microplate used in the microplate-reader. The injector apparatus may dispense reagents, which may trigger a chemical reaction resp. a luminescence reaction going along therewith in a well that is currently positioned in an optical axis of the third measurement device. The observation (the measuring resp. detecting) of a chemical reaction, e.g. by observing a luminescence reaction, may be carried out in a same well substantially simultaneously with the adding of a reagent and where required in a time interval subsequent thereto. Alternatively resp. in addition to this, the observation (the measuring resp. detecting) of a chemical reaction, e.g. by observing a luminescence reaction, may also be carried out in a well which different from the well into which the reagent is currently added. In particular, this may be carried out substantially simultaneously with the adding of the reagent into a well and where required in a time interval subsequent thereto.

It is preferred to add a reagent to a sample in a well and to simultaneously observe a chemical reaction in a well spaced at a distance thereto, e.g. in a neighboring well. In this sense, it is possible to add a reagent to a sample sequentially in each well of the microplate and to simultaneously observe a chemical reaction in a well spaced at a distance thereto, e.g. in a neighboring well e.g. by observing (measuring resp. detecting) the luminescence and/or the fluorescence and/or the absorbance of the sample contained in the well spaced at a distance, e.g. the neighboring well. By this procedure it is possible to “stimulate” one well (i.e. to add a reagent to the sample contained therein) of the microplate after the other and to observe (measure resp. detect) delayed by a time period the chemical reaction caused by the stimulation. This time period of the time delay is determined by the geometrical offset (i.e. lateral distance in an X-Y-plane) between the respective wells spaced at a distance, e.g. the respective neighboring wells, and the velocity by which the microplate and the measurement device used for observing the chemical reaction (more precisely: the optical axis thereof) are moved relative to each other, in particular in a plane that is parallel to the microplate.

This object is achieved with respect to a second aspect by the features as herein disclosed. Thereby, there is provided a method for measuring living cells in a microplate reader which comprises a housing surrounding an interior space. The method comprises the following steps:

-   a) providing a sample compartment, which is separated from an     appliance compartment and in which a holding device for     accommodating of at least one microplate is arranged,     -   a1) wherein a separating plate is arranged in the housing so as         to subdivide the interior space of the housing into the sample         compartment and the appliance compartment, or     -   a2) wherein an interior housing, in which the holding device is         arranged, is provided within the housing, and     -   wherein the separating plate or the interior housing has at         least one opening which is designed such that light emitted or         influenced by samples in wells of a microplate used or inserted         in the microplate-reader can pass therethrough from the sample         compartment to the appliance compartment, -   b) accommodating in a holding device of said microplate reader of at     least one microplate comprising wells containing samples, -   c) positioning of the samples-containing wells of this(these)     microplate(s) in relation to at least one measuring device of this     microplate reader, -   d) controlling the composition of the gas atmosphere in the sample     compartment, and -   e) using this at least one measuring device for detecting light,     -   which is emitted by samples in wells of a microplate used or         inserted in this microplate reader, and/or     -   which is influenced by samples transilluminated by light in         wells of a microplate used or inserted in this microplate         reader.

In this method, the sequence of steps, particularly steps b) to e), may be changed. Thus for example, the step d) may be executed prior to the step c) or to the step b) and/or the step e) may be executed prior to the step c) or prior to the step d).

The method may further comprise at least one or more of the following steps:

-   f) measuring light, which is influenced by samples that are     transilluminated by light in wells of a microplate used in the     microplate-reader, for measuring the absorbance of the sample, -   g) measuring light, which is emitted from samples that are     irradiated by light in wells of a microplate used in the     microplate-reader, for measuring the fluorescence of the sample, and -   h) measuring light, which is emitted from samples that are     irradiated by light in wells of a microplate used in the     microplate-reader, for measuring the luminescence of the sample.

A defined gas atmosphere may be adjusted in the sample compartment and at least one or more of the steps f), g) and h) mentioned hereinbefore may be executed with resp. under the adjusted gas atmosphere of the step d) mentioned above.

In the method, gas which is flowing into the sample compartment and/or which is present therein may be mixed and/or circulated by means of a movement device arranged in the samples compartment.

In the method, the composition of the gas atmosphere in the sample compartment may be controlled by means of a control unit. Thereby, gas may be admitted into the sample compartment through a gas inlet, wherein the gas inlet is actuated by the control unit. In the method, the proportion in the gas atmosphere in the sample compartment of at least one particular gas out of different gases may be controlled, wherein the particular gas is selected from a group, which comprises nitrogen (N₂), carbon dioxide (CO₂), oxygen (O₂), carbon monoxide (CO), hydrogen sulphide (H₂S) and sulphur dioxide (SO₂). In the method, the humidity and/or the temperature of the gas atmosphere in the sample compartment may be controlled by means of the control unit. In the method, in the sample compartment, the air may be ventilated by means of a ventilation device.

In the method for measuring living cells and/or in the use of a microplate-reader (see below), a reagent may be added to at least one sample in at least one well of the microplate by means of an injector apparatus. Thus, a chemical reaction may be caused, which can be detected by means of at least one of the measurement devices. In particular, thereby going along with the chemical reaction, a luminescence reaction resp. a change of the luminescence characteristics and/or a change in the fluorescence and/or a change in the absorbance of the sample. For example, a reagent may be added in a well, which is currently in an optical axis of the third measurement device, and may cause a chemical reaction there.

The observation (the measuring resp. detecting) of a chemical reaction, e.g. by observing the luminescence reaction caused, may be carried out in a same well substantially simultaneously with the adding of a reagent and where required in a time interval subsequent thereto. Alternatively resp. in addition to this, the observation (the measuring resp. detecting) of a chemical reaction, e.g. by observing the luminescence reaction caused, may also be carried out in a well which different from the well into which the reagent is currently added. In particular, this may be carried out substantially simultaneously with the adding of the reagent into a well and where required in a time interval subsequent thereto.

As already pointed out, it is preferred to add a reagent to a sample in a well and to simultaneously observe a chemical reaction in a well spaced at a distance thereto, e.g. in a neighboring well.

A chemical reaction in the sample (e.g. a cell culture) caused by adding a reagent resp. a cell culture may be observed resp. identified by observing (measuring resp. detecting) a kinetics of the growth resp. the change of a sample resp. the cell culture arranged in a well of a microplate. In the method, samples in wells of a microplate used in (inserted into) the microplate-reader may be transilluminated by light and/or samples in wells may be irradiated by light by means of an illumination device.

Samples in wells of the microplate used in the microplate-reader may also be irradiated by light by means of a second illumination device. The second illumination device may be arranged in the appliance compartment. It may preferably be selected from a group, which comprises a laser, a flash lamp, an LED (light emitting diode) and a laser diode. In the method, the absorbance of a sample and/or the fluorescence of a sample and/or the luminescence of a sample may be measured respectively by means of a measurement device arranged in the sample compartment or in the appliance compartment. In particular, the absorbance of the sample may be measured by means of a first measurement device arranged in the sample compartment and/or the fluorescence of the sample may be measured by means of a second measurement device arranged in the appliance compartment. Also, the luminescence of the sample may be measured by means of a third measurement device arranged in the sample compartment or in the appliance compartment.

In the microplate-reader in which the method can be performed, the control unit may comprise an O₂-sensor and/or a CO₂-sensor for controlling the oxygen content and/or carbon dioxide content of the gas atmosphere surrounding the samples-containing wells of a microplate used in (inserted into) this microplate reader. Thereby, the oxygen concentration and/or the carbon dioxide concentration of this gas atmosphere may be held at a defined value by selectively admitting carbon dioxide and/or nitrogen during the measurement of microaerophilic, optionally anaerobic and obligatorily anaerobic microorganisms, fungi or eukaryotic cells.

It is feasible in the method described above that:

-   d1) when controlling the composition of the gas atmosphere in the     sample compartment, the concentration of a gas which is already     present in the gas atmosphere, such as oxygen (O₂) or carbon dioxide     (CO₂), may be lowered by admitting a chemically inactive gas, such     as nitrogen or an inert gas, into the sample compartment (whereby in     particular the gas atmosphere including the gas which is already     present is “diluted” resp. partially squeezed out) and measuring the     concentration of the gas which is already present by means of a     sensor, such as an O₂-sensor and/or a CO₂-sensor, which is     responsive to this gas which is already present, or -   d2) when controlling the composition of the gas atmosphere in the     sample compartment, the concentration of a gas which is already     present in the gas atmosphere, such as oxygen (O₂) or carbon dioxide     (CO₂), may be raised by admitting more of this gas into the sample     compartment (whereby in particular the concentration of this gas is     selectively raised) and measuring the concentration of this gas by     means of a sensor, which is responsive to this gas, or -   d3) when controlling the composition of the gas atmosphere in the     sample compartment, the concentration of a gas which is not yet or     already present in the gas atmosphere, such as hydrogen sulphide     (H₂S) or carbon monoxide (CO), may be raised by admitting more of     this gas into the sample compartment (whereby in particular the     concentration of this gas is selectively raised) and measuring the     concentration of this gas by means of a sensor, which is responsive     to this gas.

This object mentioned above is achieved with respect to a third aspect by the use according to the invention of the aforementioned microplate reader according to the invention or by the aforementioned method according to the invention. These uses concern the measurement of living cells in a microplate reader, wherein the living cells are chosen from a group which comprises microaerophilic, optionally anaerobic and obligatorily anaerobic microorganisms, fungi and eukaryotic cells.

Additional inventive features are provided by the respective dependent claims.

The use of such a microplate reader is especially preferred in the measurement of microaerophilic or facultative anaerobic microorganisms in a defined O₂ concentration. Methods for the measurement of living cells in a microplate reader are also preferable, with the living cells being chosen from a group which comprises microaerophilic, facultative anaerobic and mandatorily anaerobic microorganisms, as well as fungi and eukaryotic cells. Multi-well plates or microtiter plates according to the ANSI-SBS Standard 2004 are designated as microplates which can comprise 6, 12, 24, 48, 96, 384 or 1536 wells for example.

The microplate reader in accordance with the invention comprises the following advantages:

-   -   The microplates with the samples to be measured need not         permanently be transferred back and forth between an incubator         and the measuring device. Such transfers can therefore be         omitted during which the cells or the cell cultures are         subjected to ambient air and can be influenced by such ambient         air in such a way that distorted measuring results will be         obtained.     -   The measurements with respect to luminescence and/or         fluorescence and/or absorbance can occur in a fully automated         manner, so that after the charging of the microplate reader in         accordance with the invention it is no longer necessary that an         operator be present.     -   In contrast to conventional (e.g. CO₂) incubators without         integrated fluorescence measurement, long-term measurements can         be performed with a microplate reader in accordance with the         invention, thus preventing the occurrence of so-called “night         windows” during which no data can be detected.     -   The control of the O₂ and/or CO₂ concentration in the atmosphere         above the wells or in the ambient environment of the wells of         microplates enable the measurement of microaerophilic,         facultative anaerobic or mandatorily anaerobic microorganisms,         fungi or eukaryotic cells under a defined O₂ and/or CO₂         concentration.

BRIEF INTRODUCTION OF THE DRAWINGS

The microplate reader in accordance with the invention and its use in accordance with the invention will now be explained by reference to the schematic illustrations which show exemplary and preferred embodiments without limiting the scope of the present invention, wherein:

FIG. 1 shows a highly schematic vertical sectional view through a microplate reader in accordance with the invention according to a preferred first embodiment, comprising an interior space of the housing which is subdivided by means of a separating plate into an appliance compartment and a sample compartment;

FIG. 2 shows a highly schematic vertical sectional view through a microplate reader in accordance with the invention according to a preferred second embodiment, comprising an interior space of the housing which is subdivided by means of a separating plate into an appliance compartment and a sample compartment;

FIG. 3 shows a highly schematic vertical sectional view through a microplate reader in accordance with the invention according to a preferred third embodiment, comprising an interior space of the housing which is subdivided by means of a separating plate into an appliance compartment and a sample compartment;

FIG. 4 shows an exemplary arrangement of an O₂ sensor, a CO₂ sensor, a fan and the gas inlet on the inside of the rear wall of a microplate reader in accordance with the invention;

FIG. 5 shows growth curves of eukaryotic tumor cells in a medium with serum (MWS) by using a microplate reader in accordance with the invention with or without CO₂ control;

FIG. 6 shows growth curves of eukaryotic tumor cells in a medium without serum (MWOS) by using a microplate reader in accordance with the invention with or without CO₂ control.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a highly schematic vertical sectional view through a microplate reader 1 in accordance with the invention according to a preferred first embodiment, comprising a housing 17 whose interior space 16 is subdivided into an appliance compartment 18 and a sample compartment 19 by means of a separating plate 15. This microplate reader 1 comprises at least one measuring device 2′, 2″, 2′″ for the detection of light.

FIG. 1 shows a first measuring device 2′ according to a first variant of the microplate reader 1 in accordance with the invention, with which light can be measured which was influenced by samples transilluminated by light in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the absorbance of the sample). This first measuring device 2′ is preferably arranged in the sample compartment 19 of the microplate reader 1 and a first illumination device 11′ for transilluminating the samples in the wells 3 of a microplate 4 is preferably arranged in the appliance compartment 18 of microplate reader 1. As a result, the first measuring device 2′ for detecting light which is influenced by samples in wells 3 of a microplate 4 used in this microplate reader 1 is arranged on a second side 14 (i.e. the bottom side) of a microplate 4 inserted in this microplate reader 1 and in the direction of a first optical axis 13′. Consequently, the first illumination device 11′ for transilluminating the samples is arranged on a first side 12 (i.e. on the upper side) of a microplate 4 inserted in this microplate reader 1 and also in the direction of this first optical axis 13′. The assignment of the first illumination device 11′ to the first optical axis 13′ occurs in this case with a first fiber-optical line 33′. The first illumination device 11′ comprises a first fiber slide 34′, a first wavelength-selective monochromator 35′ and a flash lamp 36. As a result, the light of the flash lamp 36 is guided for the transillumination of the samples by means of the first fiber-optical line 33′ in the direction of the first optical axis 13′ and via a first optical system 23′ against the samples. Said first optical system 23′ can be arranged to be height-adjustable in the direction of a Z-axis of a Cartesian system of coordinates (cf. double arrow Z). In this case, a holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 of these microplate(s) 4 in relation to the first measuring device 2′ and thereby also in relation to the first optical axis 13′ is arranged between the first illumination device 11′ and the first measuring device 2′, but preferably in the sample compartment 19.

A second measuring device 2″ is shown in FIG. 1 according to a second variant of the microplate reader 1 in accordance with the invention, with which light can be measured with respect to a second optical axis 13″, which light is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the fluorescence of the sample). Said second measuring device 2″ comprises a photo multiplier tube (PMT) 24, a second wavelength-selective monochromator 35″ and a second fiber slide 34″. For the purpose of this fluorescence measurement the first illumination device 11′ of the microplate reader 1 is used for irradiating (exciting) the samples in the wells 3 of a microplate 4. The assignment of the first illumination device 11′ to the second optical axis 13″ occurs in this case by way of a second fiber-optical line 33″ and by way of a second optical system 23″. Said second optical system 23″ can be arranged to be height-adjustable in the direction of a Z-axis of a Cartesian system of coordinates (cf. double arrow Z). As a result, the light of the flash lamp 36 is guided for irradiating the samples by means of the second fiber-optical line 33″ into the direction of the second optical axis 13″.

Two different arrangements can be used for detecting the fluorescence emitted by the samples:

-   -   In the so-called “top reading” the second optical system 23″ is         used above the microplate 4 which is connected by way of the         second fiber-optical line 33″ with the second fiber slide 34″,         so that the photo multiplier tube (PMT) 24 can be used for         detecting the fluorescence of every single sample in a well 3 of         the microplate 4.     -   In the so-called “bottom reading” there is a third optical         system 23′″ beneath the microplate 4 and is connected by way of         a third fiber-optical line 33′″ with the first and second fiber         slide 34′, 34″, so that this illumination device 11′ and the         same measuring device 2″ can be used for exciting and detecting         the fluorescence of every single sample in a well 3 of the         microplate 4.

In this case, the holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 containing the samples of these microplate(s) 4 is preferably arranged with respect to the first or second measuring device 2′, 2″ in the sample compartment 19 and beneath the first illumination device 11′ which is preferably housed in the appliance compartment 18.

A third measuring device 2′″ is shown in FIG. 1 according to a third variant of the microplate reader 1 in accordance with the invention, with which light can be measured which is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (i.e. the luminescence of the sample). A third measuring device 2′ is used for this luminescence measurement, which measuring device is preferably arranged in the appliance compartment 18 of the microplates reader 1 and with respect to a third optical axis 13′″. A generally known injector apparatus 10 which preferably reaches into the sample compartment 19 of the microplate reader 1 is used for triggering a chemical reaction which goes along with a luminescence reaction resp. a change in the luminescence characteristics, and/or a change in the fluorescence and/or a change in the absorbance in or on the samples in the wells 3 of a microplate 4. Said injector apparatus 10 is preferably configured and arranged in such a way that the reagents triggering the chemical reaction, e.g. a luminescence reaction, can be added in a well 3 of the microplate 4, when the well 3 is disposed at the time on a third optical axis 13′″ and thereby precisely above the third measuring device 2′″.

A preferred embodiment of the microplate-reader 1 is designed such that a reagent is added to a sample in a well 3 and a chemical reaction is simultaneously observed in a well spaced at a distance thereto, e.g. in a neighboring well. In this sense, it is possible to add a reagent to a sample sequentially in each well 3 of the microplate 4 and to simultaneously observe a chemical reaction in a well 3 spaced at a distance thereto, e.g. in a neighboring well e.g. by observing (measuring resp. detecting) the luminescence and/or the fluorescence and/or the absorbance of the sample contained in the well spaced at a distance, e.g. the neighboring well. By this procedure it is possible to “stimulate” one well (i.e. to add a reagent to the sample contained therein) of the microplate 4 after the other and to observe (measure resp. detect) delayed by a time period the chemical reaction caused by the stimulation. This time period of the time delay is determined by the geometrical offset (i.e. lateral distance in an X-Y-plane) between the wells 3 respectively concerned (spaced at a distance, e.g. neighboring) and the velocity by which the microplate 4 and the measurement device 2′, 2″, 2′″ (resp. the optical axis 13′, 13″, 13′″ thereof) used for observing the chemical reaction are moved relative to each other, in parallel to particular the X-Y-plane.

An according injector apparatus 10 is also conceived in the embodiments of a microplate-reader shown in the FIGS. 2 and 3, and what has been disclosed hereinbefore with respect to the injector apparatus 10 and its function resp. use applies also to the embodiments shown there.

The third measuring device 2′″ preferably comprises a fourth optical system 23′″, which can movably be arranged in the direction of a Z-axis of a Cartesian system of coordinates (cf. double arrow Z). The first, second, third and fourth optical systems 23′, 23″, 23′″ and 23″″ are arranged in an especially preferred way to be partly lowerable into the sample chamber 19 through a respective opening 20 in the separating plate 15.

A separate control unit 6′ is placed on the housing 17 of the microplate reader 1 in accordance with the invention, with the arrangement of an O₂ sensor 8, a CO₂ sensor 9, a fan 22 and a gas inlet 21 being provided on the inside of the rear wall 30 of the housing 17 of the microplate reader 1 in accordance with the invention (cf. FIG. 4). The control unit 6 of the microplate reader 1 preferably comprises an O₂ sensor 8 for measuring and controlling the oxygen content of the gas atmosphere 7 in the sample compartment 19, i.e. in the surrounding of the wells 3 containing the samples of microplates for inserted in said microplate reader 1. The control unit 6 of the microplate reader 1 comprises in an especially preferred way and alternatively to or in addition to this O₂ sensor 8 a CO₂ sensor 9 for measuring and controlling the carbon dioxide content of the gas atmosphere 7 surrounding the wells 3 containing the samples of microplate(s) 4 inserted in said microplate reader 1. The control unit 6 is therefore arranged partly within and partly without the housing 17 of the microplate reader 1 and is or can be connected by way of electrical contacts 27 and gas lines 38 with the microplate reader 1. The separate control unit 6′ is directly connected to the necessary pressure cylinders for the gas is to be used. The respective valves and throttles for the required gas connections are installed in the separate control unit 6′ (not shown). Alternatively, it is also possible to use mouse and throttles on the gas pressure cylinders, wherein these valves are preferably arranged as electrically controlled solenoid valves (of the type normally closed). FIG. 1 does not show among other things the control elements, display elements and power supplies for the separate control unit 6′ and the microplate reader 1.

FIG. 2 shows a highly schematic vertical sectional view through a microplate reader 1 in accordance with the invention according to a preferred second embodiment, comprising a housing 17 whose interior space 16 is subdivided into an appliance compartment 18 and a sample compartment 19 by means of a separating plate 15. This microplate reader 1 comprises at least one measuring device 2′, 2″, 2′″ for the detection of light.

FIG. 2 shows a first measuring device 2′ according to a first variant of the microplate reader 1 in accordance with the invention, with which light can be measured which was influenced by samples transilluminated by light in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the absorbance of the sample). This first measuring device 2′ is preferably arranged in the sample compartment 19 of the microplate reader 1 and a first illumination device 11′ for transilluminating the samples in the wells 3 of a microplate 4 is preferably arranged in the appliance compartment 18 of microplate reader 1. As a result, the first measuring device 2′ for detecting light which is influenced by samples in wells 3 of a microplate 4 used in this microplate reader 1 is arranged on a second side 14 (i.e. the bottom side) of a microplate 4 inserted in this microplate reader 1 and in the direction of a first optical axis 13′. Consequently, the first illumination device 11′ for transilluminating the samples is arranged on a first side 12 (i.e. on the upper side) of a microplate 4 inserted in this microplate reader 1 and also in the direction of this first optical axis 13′. The assignment of the first illumination device 11′ to the first optical axis 13′ occurs in this case with a partly transparent (e.g. 50%) mirror 26 or a dichroic mirror. The first illumination device 11′ comprises a first wavelength-selective filter 37′ and a flash lamp 36. As a result, the light of the flash lamp 36 is guided for the transillumination of the samples by means of the partly transparent mirror 26 in the direction of the first optical axis 13′ and via a first optical system 23′ against the samples. Said first optical system 23′ can be arranged to be height-adjustable in the direction of a Z-axis of a Cartesian system of coordinates (cf. double arrow Z). In this case, a holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 of these microplate(s) 4 in relation to the first measuring device 2′ and thereby also in relation to the first optical axis 13′ is arranged between the first illumination device 11′ and the first measuring device 2′, but preferably in the sample compartment 19.

A second measuring device 2″ is shown in FIG. 2 according to a second variant of the microplate reader 1 in accordance with the invention, with which light can be measured with respect to a first or second optical axis 13′, 13″, which light is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the fluorescence of the sample). Said second measuring device 2″ comprises a photo multiplier tube (PMT) 24 and a second wavelength-selective filter 37″. For the purpose of this fluorescence measurement the first illumination device 11′ of the microplate reader 1 is used again for irradiating (exciting) the samples in the wells 3 of a microplate 4. The assignment of the first illumination device 11′ to the second optical axis 13″ occurs in this case again by way of a partly transparent (e.g. 50%) mirror 26 or a dichroic mirror and by way of the first optical system 23′.

Two different arrangements can be used for detecting the fluorescence emitted by the samples:

-   -   In the so-called “bottom reading” there is a second optical         system 23″ beneath the microplate 4 which is connected by way of         a first fiber-optical line 33′ with the partly transparent         mirror 26 and the second filter 37″, so that the photomultiplier         tube (PMT) 24 can be used for detecting the fluorescence of         every single sample in a well 3 of the microplate 4.     -   In the so-called “top reading” the first optical system 23′ is         used above the microplate 4 which is connected by way of the         partly transparent mirror 26 with the second filter 37″, so that         the same photo multiplier tube (PMT) 24 can be used for         detecting the fluorescence of every single sample in a well 3 of         the microplate 4.

In this case, the holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 containing the samples of these microplate(s) 4 is preferably arranged with respect to the first or second measuring device 2′, 2″ in the sample compartment 19 and beneath the first illumination device 11′ which is preferably housed in the appliance compartment 18.

A third measuring device 2′″ is shown in FIG. 2 according to a third variant of the microplate reader 1 in accordance with the invention, with which light can be measured which is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (e.g. the luminescence of the sample). A third measuring device 2′ is used for this luminescence measurement, which measuring device is preferably arranged in the appliance compartment 18 of the microplates reader 1 and with respect to a third optical axis 13′″. A generally known injector apparatus 10 which preferably reaches into the sample compartment 19 of the microplate reader 1 is used for triggering a chemical reaction, with which there may go along a luminescence reaction resp. a change in the luminescence characteristics, and/or a change in the fluorescence and/or a change in the absorbance in or on the samples in the wells 3 of a microplate 4. Said injector apparatus 10 is preferably configured and arranged in such a way that the reagents triggering the chemical reaction, e.g. a luminescence reaction, can be added in a well 3 of the microplate 4, when the well 3 is disposed at the time on the third optical axis 13′″ and thereby precisely above the third measuring device 2′″. The third measuring device 2′″ preferably comprises a third optical system 23′″ which can movably be arranged in the direction of a Z-axis of a Cartesian system of coordinates (cf. double arrow Z). The first and second optical systems 23′, 23″ are arranged in an especially preferred way to be partly lowerable into the sample chamber 19 through a respective opening 20 in the separating plate 15.

The same control unit 6 is preferably used in all aforementioned variants of the microplate reader 1 in accordance with the invention.

FIG. 3 shows a highly schematic vertical sectional view through a microplate reader 1 in accordance with the invention according to a preferred third embodiment, comprising a housing 17 whose interior space 16 is subdivided into an appliance compartment 18 and a sample compartment 19 by means of an interior housing 39. This microplate reader 1 comprises at least one measuring device 2′, 2″, 2′″ for the detection of light. The interior housing 39 comprises an opening 20, which is designed such that light emitted or influenced by samples in wells 3 of a microplate 4 used in the microplate-reader 1 may pass therethrough from the samples compartment 19 into the appliance compartment 18.

FIG. 3 shows a first measuring device 2′ according to a first variant of the microplate reader 1 in accordance with the invention, with which light can be measured which was influenced by samples transilluminated by light in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the absorbance of the sample). This first measuring device 2′ is preferably arranged in the appliance compartment 18 of the microplate reader 1 and a first illumination device 11′ for transilluminating the samples in the wells 3 of a microplate 4 is preferably arranged in the sample compartment of the microplate reader 1. As a result, the first measuring device 2′ for detecting light which is influenced by samples in wells 3 of a microplate 4 used in this microplate reader 1 is arranged on a first side 12 (i.e. the upper side) of a microplate 4 inserted in this microplate reader 1 and in the direction of a first optical axis 13′. Consequently, the first illumination device 11′ for transilluminating the samples is arranged on a second side 14 (i.e. on the bottom side) of a microplate 4 inserted in this microplate reader 1 and also in the direction of this first optical axis 13′. The first measuring device 2′ preferably comprises a mirror 25 for deflecting the light coming from the samples in the direction of a photo multiplier tube or PMT 24. Alternatively, the light coming from the samples can be supplied by means of the fiber optics of the PMT 24 (not shown). Similarly, the first illumination device 11′ can be arranged as a lamp and be arranged on the first optical axis 13′. Alternatively, the light for transilluminating the samples can be guided by means of mirrors or fiber optics (both not shown) in the direction of the first optical axis 13′. In this case, a holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 containing the samples of these microplate(s) 4 is preferably arranged with respect to the first measuring device 2′ between the first illumination device 11′ and the first measuring device 2′, but preferably in the sample compartment 19.

A second measuring device 2″ is shown in FIG. 3 according to a second variant of the microplate reader 1 in accordance with the invention, with which light can be measured which is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (therefore the fluorescence of the sample). For the purpose of this fluorescence measurement the first measuring device 2′ of the microplate reader 1 is used again which is identical in this case with the second measuring device 2″. A second illumination device 11″ (preferably a laser, a flash lamp or a laser diode) is used as a source for the required exciting light for irradiating the samples in the wells 3 of a microplate 4. Said second illumination device 11″ is preferably arranged in the housing space 18 of the microplate reader 1. As a result, both the first measuring device 2′ for the detection of light which is emitted by samples in wells 3 of a microplate 4 inserted in this microplate reader 1 and also the second illumination device 11″ are disposed on the same side 12 (therefore on the upper side) of a microplate 4 inserted in this microplate reader 1. The second illumination device 11″ preferably comprises a partly (e.g. 50%) transparent mirror 26 or a dichroic mirror for deflecting the exciting light in the direction of the first optical axis 13′ and therefore onto or into the samples. In this case, the holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 containing the samples of these microplate(s) 4 is preferably arranged with respect to the first measuring device 2′ in the sample compartment 19 and beneath the second illumination device 11″ and the first measuring device 2′ which are preferably housed in the appliance compartment 18.

A third measuring device 2′″ is shown in FIG. 3 according to a third variant of the microplate reader 1 in accordance with the invention, with which light can be measured which is emitted by samples in wells 3 of a microplate 4 used in this microplate reader 1 (i.e. the luminescence of the sample). A third measuring device 2′″ is used for this luminescence measurement, which third measuring device is preferably arranged in the sample compartment 19 of the microplate reader 1 and with respect to a second optical axis 13″. A generally known injector apparatus 10 which is preferably arranged in the sample compartment 19 of the microplate reader 1 is used for triggering a chemical reaction, with which there may go along a luminescence reaction resp. a change in the luminescence characteristics, a change in the fluorescence and/or a change in the absorbance in or on the samples in the wells 3 of a microplate 4. Said injector apparatus 10 is for example configured and arranged in such a way that the reagents triggering the chemical reaction, e.g. a luminescent reaction, can be added in a well 3 of the microplate 4, when the well 3 is disposed at the time on the second optical axis 13″ and thereby precisely above the third measuring device 2′″. In this case, the holding device 5 for accommodating at least one microplate 4 and for positioning the wells 3 containing the samples of these microplate(s) 4 is preferably arranged with respect to the second measuring device 2″ in the sample compartment 19, beneath the injector apparatus 10 and above the third measuring device 2′″. The first measuring device 2′ preferably comprises a first optical system 23′ which can movably be arranged in the direction of a Z-axis of a Cartesian system of coordinates. The first optical system 23′ is arranged in an especially preferred way to be partly lowerable into the sample chamber 19 through a respective opening 20 in the interior housing 39, according to the first embodiment as shown in FIG. 3.

The microplate 4 resp. the holding device 5 is movable in the direction of a Z-axis of a Cartesian coordinate system (cf. double arrow Z in FIGS. 2 and 3) relative to the first, second and/or third optical system 23′, 23″, 23′″, respectively, of the first, second and/or third measurement device 2′, 2″, 2′″. The microplate 4 resp. the holding device 5 (cf. double arrow X, Y) and/or the first, second and/or third measurement device 2′, 2″, 2′″ resp. their respective first, second and/or third optical system 23′, 23″, 23′″ may also be designed to be movable in the direction of an X-axis and/or a Y-axis of a Cartesian coordinate system. This applies for all embodiments shown and variants of the microplate-reader 1 according to the invention and possible modifications thereof.

All embodiments and variants of the microplate reader 1 in accordance with the invention are characterized in that the microplate reader 1 comprises a control unit 6 for controlling the composition of a gas atmosphere 7 surrounding the wells 3 containing the samples of microplates 4 inserted in this microplate reader 1. Moreover, the holding device 5 is arranged to be movable preferably in the direction of an X-axis and Y-axis of a Cartesian system of coordinates (cf. FIGS. 1 to 3: double arrows X, Y).

FIG. 4 shows an exemplary arrangement of an O₂ sensor 8, a CO₂ sensor 9, a fan 22 and a gas inlet 21 on the inside of the rear wall 30 of the housing 17 of a microplate reader 1 in accordance with the invention. The control unit 6 of the microplate reader 1 preferably comprises an O₂ sensor 8 for measuring and controlling the oxygen content of the gas atmosphere 7 surrounding the wells 3 containing the samples of the microplates 4 inserted in this microplate reader 1. The control unit 6 of the microplate reader 1 comprises in an especially preferred way a CO₂ sensor 9 alternatively to or in addition to said O₂ sensor 8 for measuring and controlling the carbon dioxide content of the gas atmosphere 7 surrounding the wells 3 containing the samples of microplate(s) 4 inserted in this microplate reader 1. This control unit 6 can be arranged partly outside of the housing 17 of the microplate reader 1 and can be connected or connectable for example by way of electric contacts 27 and gas conduits (not shown) with the microplate reader 1 (not shown). It can also be provided however that the control unit 6 is integrated completely in the microplate reader 1 and is housed in a common housing 17 (cf. FIGS. 2 and 3). In this case, the microplate reader 1 would be connected directly to the necessary pressure cylinders for the gases to be used (cf. FIGS. 2 and 3). Preferably, the respective valves and throttles are then also installed in the housing 17 of the microplate reader 1 (not shown). Alternatively, valves and throttles on the gas pressure cylinders can also be used, with these valves preferably being arranged as electrically controlled solenoid valves (of the type normally closed) (not shown).

The control unit preferably comprises a computer 28 with respective software. In this case, the computer 28 can be connected with a central computer 29 of the microplate reader 1 (cf. FIG. 1) (housed in a separate housing 17′ for example) or be integrated in this central computer 29 of the microplate reader 1 (not shown).

Preferably, the nitrogen (N₂) and/or the carbon dioxide (CO₂) to be used for displacing the ambient air are provided in pressure cylinders, with generally known control valves and throttles be used in these pressure cylinders in order to set the required feed pressure for these gases. Such process gases can alternatively also be obtained from other sources (e.g. from in-house conduits). In addition to nitrogen and carbon dioxide, other gases can be used for producing a defined percentage in the atmosphere usually prevailing in the sample compartment (combined with respective gas detectors in the sample compartment 19). By observing the potentially applicable safety measures it is therefore also possible to introduce other gases such as noble gases or inert gases (e.g. argon) or also reactive or poisonous gases (e.g. oxygen, carbon monoxide, hydrogen sulfide or sulfur dioxide) into the sample compartment 19 of the microplate reader 1 by way of a gas inlet 21 via a microplate 4 inserted in the area of the wells 3 in said microplate reader 1 for generating a specific composition of the gas atmosphere. Preferably, the control unit 6 is equipped with sufficient gas conduits and control valves so that even more complex gas compositions with several gas components are enabled.

With respect to suitable CO₂ sensors, the currently used CO₂ sensor shall be mentioned: SenseAir® CO₂ Engine® ICB, Part No.: 033-9-0001 of SenseAir AB in SE-820 60 Delsbo, Sweden. With respect to suitable O₂ sensors, the currently used O₂ sensor shall be mentioned: Pewatron FCX-MEP2-F-CH oxygen module of Pewatron AG in CH-8052 Zurich, Switzerland.

The control unit 6 in combination with an O₂ sensor allows controlling the oxygen content of the gas atmosphere 7 surrounding the wells 3 containing the samples of microplate(s) 4 inserted in this microplate reader 1, so that microplate reader 1 can be used in the measurement of microaerophilic or facultative anaerobic microorganisms under a defined O₂ concentration. The measurement of anaerobic microorganisms or eukaryotic cells under a defined O₂ concentration is thus enabled.

The control unit 6 in combination with a CO₂ sensor allows controlling the carbon dioxide content of the gas atmosphere 7 surrounding the wells 3 containing the samples of microplate(s) 4 inserted in this microplate reader 1, so that this microplate reader 1 can be used in the measurement of living cell cultures under a defined CO₂ concentration.

In connection with the present invention, cell cultures, biological cell accumulations separated from such cell cultures or obtained otherwise, or individual cells will be designated as cells, with such cells comprising microorganisms and fungi and animal and plant eukaryotic cells.

Any combination of the elements of the microplate reader 1 and the control unit 6 in accordance with the invention as shown in FIGS. 1 to 4 shall belong to the scope of the present invention.

The 5% CO₂ regulation of eukaryotic tumor cells was tested with a microplate reader 1 of the first embodiment in accordance with the invention (cf. FIG. 1). GFP-transfected A431 cells were used for fluorescence determination. The abbreviation GFP designates the generally known “green fluorescent protein” with excitation maximum at a wavelength of 396 nm and 475 nm, and with an emission maximum at a wavelength of 508 nm. In order to record the respective growth curves over 72 hours, these cells were suspended in media with 10% serum (MWS) or in media without serum (MWOS).

The medium with serum (MWS) had the following composition: Dulbecco's modified Eagle's Medium (DMEM containing phenol red) with 4.5 g/l glucose, supplemented by 10 mM of HEPES buffer, 2 mM of L-glutamine, 1 mM of Na-pyruvate, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin and 5% (v/v) of fetal bovine serum (FBS). All media components were obtained from PAA laboratories, Linz, Austria.

The medium without serum (MWOS) contained the same components, but no fetal bovine serum (FBS).

Microplates 4 of type Greiner Standard Cell Culture MTP 96 Well sterile coating (Greiner 96 well, black, flat & clear bottom, sterile—culture tissue plates) were used. 48 wells 3 of these microplates 4 were respectively charged with 2500 A431GFP cells and with 10% serum (sample type A, cf. FIG. 5). The remaining 48 wells of these microplates 4 were respectively charged with 2500 A431GFP cells without serum (sample type B, cf. FIG. 6). Two test series were performed with these 2 sample types A and B:

The first test series consisted of measuring the CO₂ concentration in the sample compartment 19 with a CO₂ sensor (SenseAir®, Typ CO₂ Engine® ICB, Part No.: 033-9-0001), which concentration was kept constantly at 5% with a control unit 6 with an integrated electronic control system. This was achieved in such a way that the atmosphere in the sample compartment 19 and in the appliance compartment 18 of the microplate reader 1 in accordance with the invention initially had a gas composition which corresponded to the one of the ambient air, i.e. it corresponded approximately to the standard atmosphere, which has the following composition: oxygen 20.93%, nitrogen 78.10%, argon 0.93%, carbon dioxide 0.03%, hydrogen, neon, helium, krypton and xenon 0.01%. The housing 17 of the microplate reader 1 was closed after the positioning of the microplate 4 on the holding device 5 of the microplate reader 1 and CO₂ gas was introduced into the sample compartment 19 via the gas inlet 21 until the CO₂ gas concentration reached 5%. This CO₂ concentration was held permanently over the duration of the measurement in that CO₂ gas was introduced into the sample compartment 19 as required (once the CO₂ sensor determined an inadequate carbon dioxide concentration). The gas mixture in the sample compartment 19 was circulated constantly with the fan 22. The temperature was kept constant at 37° C. in any case.

A second test series consisted of not introducing any additional CO₂ into the sample compartment 19 and thereby subjecting the samples permanently to ambient air. The temperature was kept constant at 37° in any case.

The fluorescence of each sample (each well 3) was respectively excited with a first illumination device 11′ which was arranged beneath the microplate 4 (excitation at λ=485 nm; detection of fluorescence at λ=535 nm) and guided by means of the second optical system 23″ to a photo multiplier tube 24 for detection. The individual fluorescence of the individual samples was measured depending on the time. The individual intensities of the fluorescence of every single well detected by the photo multiplier tube 24 were processed in the central computer 29 of the microplate reader 1 and then shown in curve diagrams (cf. FIGS. 5 and 6).

FIG. 5 shows growth curves of eukaryotic tumor cells in a medium with 10% serum (MWS) produced with or without CO₂ control by using a microplate reader 1 in accordance with the invention. The reference numeral 31 designates the sam-ples in which the CO₂ concentration was kept permanently at 5%, and reference numerals 32 designates the samples which were subjected to normal ambient air. FIG. 5 shows that the cells acted approximately similarly during the first 35 hours, with cell vitality (measured on the basis of the corresponding GFP-signal (i.e. on the basis of intensity of fluorescence)) of the samples 31 in which the CO₂ concen-tration was kept permanently at 5% being permanently slightly lower than the cell vitality of the samples 32 which were subjected to normal ambient air. After ap-proximately 35 hours, growth of the cells of the samples 32 which were subjected to normal ambient air slowed down dramatically. It reached a maximum of approximately 450% after approximately 60 hours and decreased thereafter to beneath 400% of the initial value at the end of the measurement at 75 hours. In contrast thereto, the cell vitality of the samples 31 in which the CO₂ concentration was kept permanently at 5% increased virtually linearly after 35 hours of measurement duration and reached approximately 900% of the initial value up until the end of the measurement at 75 hours.

FIG. 6 shows growth curves of eukaryotic tumor cells in a medium without 10% serum (MWOS) produced with or without CO₂ control by using a microplate reader 1 in accordance with the invention. The reference numeral 31 designates the samples in which the CO₂ concentration was kept permanently at 5%, and reference numerals 32 designates the samples which were subjected to normal ambient air. FIG. 6 shows that the cells acted approximately similarly during the first 35 hours, with cell vitality (measured on the basis of the corresponding GFP-signal (i.e. on the basis of intensity of fluorescence)) of the samples 31 in which the CO₂ concentration was kept permanently at 5% being permanently slightly lower than the cell vitality of the samples 32 which were subjected to normal ambient air. After approximately 35 hours, growth of the cells of the samples 32 which were subjected to normal ambient air reached a maximum of 200% and dropped to approximately the initial value of 100% up until the end of the measurement at 75 hours. In contrast thereto, the cell vitality of the samples 31 in which the CO₂ concentration was kept permanently at 5% increased in virtually the same amount even after approximately 35 hours of measurement duration and reached approximately 300% of the initial value up until the end of the measurement at 75 hours.

The illustrated results show that the cells held permanently under a CO₂ concentration of 5% are able to show substantially higher cell vitality than cells held under normal ambient air. This larger cell vitality plays a role especially after an incubation period of more than 40 hours. The further addition of 10% serum increases cell vitality during the first 35 hours approximately by a factor of 2, and even by a factor of 3 after 75 hours.

Such uninterruptible long-term studies actually become possible with the current invention, unlike conventional CO₂ incubators without integrated fluorescent measurement where typically a night window of approximately 14 hours during which no data can be collected needs to be taken into account.

No microplate readers are known from the state of the art which, comprise sensors and a control unit for controlling the composition of the gas atmosphere above the wells or in the ambient environment of the wells containing the samples of a microplate used in this microplate reader.

Although the German published application DE 10 2005 033 927 A1 discloses an illumination device for transmitted light contrast in the bright field for inverse microscopes for observing living cells, this illumination device is integrated in a hermetically sealed, light-proof and compact incubator of the inverse microscopes in such a way that each individual well of a sample holder arranged as a microtiter plate is illuminated successively and that simultaneously thermal stabilization of the sample volume is ensured over a prolonged period of time in combination with the lowest possible heat losses. This illumination device is arranged in a stationary manner in an upper part of the incubator and above a projection lens. An inserted microtiter plate, Petri dish or the like can be moved relative to the projection lens on a microscope stage arranged in a bottom part of the incubator. The incubator comprises a first in a chamber which is enclosed completely by an outer second chamber and is thereby thermally decoupled from the ambient environment. The microtiter plate with the samples is disposed in said inner chamber in which the temperature, the air humidity and the CO₂ content are controllable in a controlled manner by control devices. This German published application DE 10 2005 033 927 A1 relates exclusively to inverse microscopes, the configuration and use of which differ considerably from a microplate reader which does not comprise any imaging function and enables substantially shorter measuring periods per well. Microplate readers and especially O₂ sensors are not mentioned in this publication.

A climatic chamber for observing samples in microtiter plates is also known from the patent EP 1 575 706 B1. A conditioning stream of medium in the form of air with a defined air humidity and/or temperature can be introduced into this climatic chamber. The defined introduction of CO₂ gas and the arrangement of a temperature sensor, humidity sensor and/or gas sensor close to the sample holder carrying the microtiter plate can be provided. Microplate reader and especially O₂ sensors are not mentioned in this publication.

As a result, the state of the art does not make obvious to the person skilled in the art the microplate reader 1 in accordance with the invention with the control unit 6 for controlling the composition of a gas atmosphere 7 above or in the ambient environment of the wells 3 containing the samples of microplates 4 used in this microplate reader 1.

Further preferred applications of the microplate reader 1 and the control unit 6 in accordance with the invention relate for example to examinations with respect to the influence of O₂ partial pressure on the growth of microorganisms such as Rhodospirillum rubrum [Biedermann et al. 1967, “Archiv für Mikrobiologie” (Archive for Microbiology) 56, 133-147] and examinations on microaerophilic alginate production, in which preferably an O₂ concentration of 2.5-5% (preferably 2.5%) is to be set [Wael Sabra 1999: Microaerophilic alginate production with Azotobacter vinelandii; Dissertation at the Common Scientific Faculty of the Braunschweig University of Technology].

Same or corresponding features of the microplate-reader 1 shown in the Figures are provided with same reference numerals, also when this is not expressly referred to in the description. Also, arbitrary combinations of features of embodiments shown the Figures resp. of technical equivalents of these features belong to the scope of the invention as herein disclosed.

List of reference numerals:  1 Microplate reader  2′ First measuring device  2″ Second measuring device  2′″ Third measuring device  3 Wells  4 Microplate  5 Holding device  6 Control unit  6′ Separate control unit  7 Gas atmosphere  8 O₂ sensor  9 CO₂ sensor 10 Injector apparatus 11′ First illumination device 11″ Second illumination device 12 First side of an inserted microplate 13′ First optical axis 13″ Second optical axis 13′″ Third optical axis 14 Second side of an inserted microplate 15 Separating plate 16 Interior space 17 Housing of the reader 17′ Separate housing 18 Appliance compartment 19 Sample compartment 20 Opening 21 Gas inlet 22 Fan 23′ First optical system 23″ Second optical system 23′″ Third optical system 24 PMT photo multiplier tube 25 Mirror 26 Partially transparent mirror 27 Electrical contacts 28 Computer 29 Central computer of microplate reader 30 Rear wall 31 Growth curve of cells with CO₂ control 32 Growth curve of cells without CO₂ control 33′ First fiber-optical line 33″ Second fiber-optical line 33′″ Third fiber-optical line 34′ First fiber slide 34″ Second fiber slide 35′ First monochromator 35″ Second monochromator 36 Flash lamp 37′ First filter 37″ Second filter 38 Gas lines 39 interior housing 

What is claimed is:
 1. A microplate reader (1), comprising: a) at least one measuring device (2′,2″,2′″/which is selected from a group comprising: a1) a first measurement device (2′) which is designed for measuring the absorbance of samples in wells (3) of a microplate (4) used resp. inserted in the microplate-reader (1), a2) a second measurement device (2″) which is designed for measuring the fluorescence of samples that are irradiated with light in wells (3) of a microplate (4) used resp. inserted in the microplate-reader (1), and a3) a third measurement device (2′″) which is designed for measuring the luminescence of samples in wells (3) of a microplate (4) used resp. inserted in the microplate-reader (1); b) a holding device (5) for accommodating at least one microplate (4) and for positioning the samples-containing wells (3) of these microplate(s) (4) in relation to the at least one measuring device (2′,2″,2′″); c) a control unit (6,6′) for controlling the composition of a gas atmosphere (7) surrounding the samples-containing wells (3) of the microplates (4) used resp. inserted in this microplate reader (1); wherein the microplate-reader (1) further comprises d) a zoning means alternatively configured as: d1) a separating plate (15) which subdivides an interior space (16) of a housing (17) of the microplate-reader (1) into an appliance compartment (18) and a sample compartment (19) and which comprises at least one opening (20), which separating plate (15) is designed such that light which is irradiated or influenced by samples in wells (3) of a microplate (4) used in the microplate-reader (1) can pass therethrough from the samples compartment (19) to the appliance compartment (18), or d2) an interior housing (39), which is arranged within a housing (17) of the microplate-reader (1), within which the holding device (5) is arranged, which interior housing (39) subdivides an interior space (16) of the housing (17) of the microplate-reader (1) into an appliance compartment (18) and a sample compartment (19) surrounding the holding device (5), and which comprises at least one opening (20), which is designed such that light which is irradiated or influenced by samples in wells (3) of a microplate (4) used in the microplate-reader (1) can pass therethrough from the samples compartment (19) to the appliance compartment (18).
 2. A microplate reader (1) according to claim 1, characterized in that the sample compartment (19) is separated from the appliance compartment (18) substantially light-tight and substantially gas-tight by means of the separating plate (15) or the interior housing (39), preferably a proofing device extending around the opening being provided.
 3. A microplate reader (1) according to claim 1, characterized in that a movement device for mixing and/or circulating gas that is present in and/or flowing into the sample compartment (19) is arranged in the sample compartment (19).
 4. A microplate reader (1) according to claim 3, characterized in that the movement device comprises at least one of the following devices: a blower (22), a device comprising one or more baffle plates, an agitation device comprising one or more paddles, or a structured jet nozzle system.
 5. A microplate reader (1) according to claim 1, characterized in that the control unit (6,6′) comprises a computer (28) that comprises corresponding software, wherein said computer (28) is configured to be connectable to a central computer (29) of the microplate-reader (1) or integrated therein.
 6. A microplate reader (1) according to claim 1, characterized by a gas inlet (21) which is configured to be actuatable by the control unit (6, 6′) and for admitting gas into the sample compartment (19).
 7. A microplate reader (1) according to claim 1, characterized in that the control unit (6,6′) comprises gas sensors for measuring different gases in the sample compartment (19) and for controlling the composition of the gas atmosphere (7) surrounding the samples-containing wells (3) of a microplate (4) used in this microplate-reader (1).
 8. A microplate reader (1) according to claim 7, characterized in that the different gases comprise nitrogen (N₂), carbon dioxide (CO₂), oxygen (O₂), carbon monoxide (CO), hydrogen sulphide (H₂S) and/or sulphur dioxide (SO₂).
 9. A microplate reader (1) according to claim 1, characterized in that the control unit (6,6′) comprises, in the sample compartment, a humidity sensor for measuring the humidity of the gas atmosphere (7) and a temperature sensor for measuring the temperature of the gas atmosphere (7).
 10. A microplate reader (1) according to claim 1, characterized by at least one illumination device (11′, 11″) designed for transilluminating resp. irradiating with light samples in wells (3) of a microplate (4) used in the microplate-reader (1).
 11. A microplate reader (1) according to claim 1, characterized in that the first measurement device (2′), the second measurement device (2″) and/or the third measurement device (2′″) respectively comprise a light guide having an admission point for light and an exit point for light, and a light detector device arranged for measuring light exiting from the exit point, and in that the first accession point for light into the first measurement device (2′), the second accession point for light into the second measurement device (2″) and the third accession point for light into the third measurement device (2′″) are respectively arranged in the sample compartment (19).
 12. A microplate reader (1) according to claim 11, characterized in that the first light detector device of the first measurement device (2′) and/or the third light detector device of the third measurement device (2′″) are arranged in the sample compartment (19).
 13. A microplate reader (1) according to claim 1, characterized in that the first, second and/or third measuring device (2′,2″,2′″) respectively comprises a first, second and/or third optical system (23′,23″,23″), wherein at least one of these optical systems (23′,23″,23′″) and/or the holding device (5) are designed to be movable relative to each other in the direction of a Z-axis of a Cartesian coordinate system.
 14. A microplate reader (1) according to claim 1, characterized by an injector apparatus (10) designed for adding a reagent to the samples in wells (3) of a microplate (4) used in the microplate-reader (1).
 15. A method for measuring living cells in a microplate reader (1) comprising a housing (17) surrounding an interior space (16), wherein the method comprises the following steps: a) providing a sample compartment (19), which is separated from an appliance compartment (18) and in which a holding device (5) for accommodating of at least one microplate (4) is arranged, a1) wherein a separating plate (15) is arranged in the housing (17) so as to subdivide the interior space (16) of the housing (16) into the sample compartment (19) and the appliance compartment (19), or a2) wherein an interior housing (39), in which the holding device (5) is arranged, is provided within the housing (17), and wherein the separating plate (15) resp. the interior housing (39) has at least one opening (20) which is designed such that light emitted or influenced by samples in wells (3) of a microplate (4) used resp. inserted in the microplate-reader (1) can pass therethrough from the sample compartment (19) to the appliance compartment (18), b) accommodating in a holding device (5) of this microplate reader (1) at least one microplate (4) comprising wells (3) containing the samples; c) positioning the samples-containing wells (3) of this(these) microplate(s) (4) in relation to at least one measuring device (2′,2″,2′″) of this microplate reader (1), d) controlling the composition of the gas atmosphere (7) in the sample compartment (19), and e) using at least one measuring device (2′,2″,2′″) for detecting light which is emitted by samples in wells (3) of a microplate (4) used resp. inserted in this microplate reader, and/or which is influenced by samples transilluminated by light in wells (3) of a microplate (4) used resp. inserted in this microplate reader (1).
 16. Method according to claim 15, further characterized by at least one or more of the following steps: f) measuring light, which is influenced by samples that are transilluminated by light in wells (3) of a microplate (4) used in the microplate-reader (1), for measuring the absorbance of the sample, g) measuring light, which is emitted from samples that are irradiated by light in wells (3) of a microplate (4) used in the microplate-reader (1), for measuring the fluorescence of the sample, and h) measuring light, which is emitted from samples that are irradiated by light in wells (3) of a microplate (4) used in the microplate-reader (1), for measuring the luminescence of the sample.
 17. Method according to claim 16, characterized in that a defined gas atmosphere is adjusted in the sample compartment (19) and at least one or more of the steps f), g) and h) is executed with resp. under the adjusted gas atmosphere of the step d).
 18. Method according to claim 15, characterized by mixing and/or circulating gas which is flowing into the sample compartment (19) and/or which is present therein by means of a movement device arranged in the samples compartment (19).
 19. Method according to claim 15, characterized by controlling the composition of the gas atmosphere (7) in the sample compartment (19) by means of a control unit (6,6′).
 20. Method according to claim 19, characterized by admitting gas into the sample compartment (19) through a gas inlet (21), which is actuated by the control unit (6,6′).
 21. Method according to claim 19, characterized by controlling the proportion in the gas atmosphere (7) in the sample compartment (19) of at least one particular gas out of different gases, wherein the particular gas is selected from a group, which comprises nitrogen (N₂), carbon dioxide (CO₂), oxygen (O₂), carbon monoxide (CO), hydrogen sulphide (H₂S) and sulphur dioxide (SO₂).
 22. Method according to claim 19, characterized by controlling the humidity and/or the temperature of the gas atmosphere (7) in the sample compartment (19) by means of the control unit (6,6′).
 23. Method according to claim 15, characterized by adding a reagent to at least one sample in at least one well (4) of the microplate (4) by means of an injector apparatus (10), and thus causing a chemical reaction which can be detected by means of at least one of the measurement devices (2′,2″,2′″).
 24. Method according to claim 15, characterized by transilluminating samples in wells (3) of a microplate (4) used in the microplate-reader (1) by light and/or irradiating samples in wells by light by means of an illumination device (11′,11″).
 25. Method according to claim 15, characterized by measuring the absorbance of a sample and/or the fluorescence of a sample and/or the luminescence of a sample respectively by means of a measurement device (2′,2″,2′″) arranged in the sample compartment (19) or in the appliance compartment (18).
 26. Method according to claim 15, characterized in that the control unit (6,6′) comprises an O₂-sensor and/or a CO₂ sensor for controlling the oxygen and/or carbon dioxide content of the gas atmosphere (7) surrounding the samples-containing wells (3) of the microplate(s) (4) used in this microplate reader (1), wherein the O₂ and/or the CO₂ concentration of this gas atmosphere is held at a defined value by purposefully introducing carbon dioxide and/or nitrogen during the measurement of microaerophilic, optionally anaerobic or obligatorily anaerobic microorganisms, fungi or eukaryotic cells.
 27. Method according to claim 15, characterized in that d1) when controlling the composition of the gas atmosphere (7) in the sample compartment (19), the concentration of a gas which is already present in the gas atmosphere (7), such as oxygen (O₂) or carbon dioxide (CO₂), is lowered by admitting a chemically inactive gas, such as nitrogen or an inert gas, into the sample compartment (19) and the concentration of the gas which is already present is measured by means of a sensor, such as an O₂-sensor and/or a CO₂-sensor, which is responsive to this gas which is already present, or in that d2) when controlling the composition of the gas atmosphere (7) in the sample compartment (19), the concentration of a gas which is already present in the gas atmosphere (7), such as oxygen (O₂) or carbon dioxide (CO₂), is raised by admitting more of this gas into the sample compartment (19) and the concentration of this gas is measured by means of a sensor, which is responsive to this gas, or in that d3) when controlling the composition of the gas atmosphere (7) in the sample compartment (19), the concentration of a gas which is not yet or already present in the gas atmosphere (7), such as hydrogen sulphide (H₂S) or carbon monoxide (CO), is raised by admitting more of this gas into the sample compartment (19) and the concentration of this gas is measured by means of a sensor, which is responsive to this gas.
 28. Method of utilizing a microplate reader (1) according to claim 1, wherein living cells are measured in a controlled gas atmosphere (7), wherein the living cells are chosen from a group which comprises microaerophilic, optionally anaerobic and obligatorily anaerobic microorganisms as well as fungi and eukaryotic cells.
 29. Method for measuring living cells in a microplate reader (1) according to claim 15, wherein living cells are measured in a controlled gas atmosphere (7), wherein the living cells are chosen from a group which comprises microaerophilic, optionally anaerobic and obligatorily anaerobic microorganisms as well as fungi and eukaryotic cells. 